Caenorhabditis elegans TEN-1 is essential for basement membrane function
INAUGURALDISSERTATION
zur
Erlangung der Würde eines Doktors der Philosophie vorgelegt der
Philosophisch – Naturwissenschaftlichen Fakultät der Universität Basel
von
Agnieszka Trzebiatowska
aus Szczecin, Polen
Genehmigt von der Philosophisch – Naturwissenschaftlichen Fakultät auf Antrag von Prof. Ruth Chiquet-Ehrismann and Prof. Markus Affolter.
Basel, den 19.02.2008
Prof. Dr. Hans-Peter Hauri
Dekan
Acknowledgements
First of all, I would like to thank my supervisor, Prof. Ruth Chiquet-Ehrismann, for giving me the opportunity to work in her lab, for her continuous support and guidance.
I would also like to thank the C. elegans members of our lab, Krzysztof Drabikowski, Stefano Canevascini, Jacqueline Ferralli and Ulrike Topf, for an introduction into the C. elegans world, their discussion and technical support. I am very grateful to present and past members of our lab for their help and friendly atmosphere. I would also like to thank Ursula Sauder from ZMB of the University of Basel for a fruitful collaboration and the C. elegans colleagues at the FMI for critical discussion.
I would like to thank Prof. Markus Affolter and Dr. Krzysztof Drabikowski for their scientific input as members of my thesis committee.
Finally, I am particularly grateful to my family for their continuous support and understanding.
Table of contents
I. Summary... 7
II. Introduction ... 8
II.1. Teneurins... 8
II.1.1. Protein domains and structure ... 8
II.1.2. Teneurin expression patterns... 10
II.1.3. Teneurin function – knockout studies ... 11
II.1.4. Teneurin processing ... 12
II.1.5. Teneurin interacting proteins ... 13
II.2. Caenorhabditis elegans as a model system... 14
II.2.1. Basement membranes and their receptors... 15
II.2.2. Epithelial morphogenesis ... 20
II.2.3. Pharynx development ... 22
II.2.4. Gonad development... 25
II.3. Aim of the work ... 28
III. Results... 29
III.1. Results – published ... 29
III.1.1. Teneurins – proteins with fundamental roles in development ... 29
III.1.2. ten-1, an essential gene for germ cell development, epidermal morphogenesis, gonad migration, and neuronal pathfinding in Caenorhabditis elegans ... 36
III.2. Results – submitted ... 49
III.2.1. C. elegans teneurin, ten-1, is required for gonadal and pharyngeal basement membrane integrity and acts redundantly with integrin ina-1 and dystroglycan dgn-1 ... 49
III.3. Results – unpublished ... 80
III.3.1. Expression of truncated ten-1 transcripts in tm651 and ok641 mutants ... 80
III.3.2. Somatic gonad disorganization in ten-1 mutant worms... 81
III.3.3. Basement membrane ultrastructure in the ten-1 mutant worms ... 82
TABLE OF CONTENTS
III.3.6. Does teneurin-2 bind laminin?... 90
IV. Discussion... 92
IV.1. Our aspiration ... 92
IV.2. ten-1 acts redundantly with genes encoding basement membrane proteins and receptors... 93
IV.3. TEN-1 is essential for basement membrane maintenance ... 93
IV.4. Localization of TEN-1 ... 94
IV.5. Role of TEN-1 domains and isoforms ... 94
IV.6. TEN-1 function in somatic gonad cells ... 95
IV.7. ten-1 acts redundantly with dgn-1, nid-1 and ina-1 in pharyngeal and hypodermal morphogenesis ... 97
IV.8. EPI-1 is a potential basement membrane ligand for TEN-1... 98
IV.9. TEN-1 signaling... 99
V. Appendix... 100
V.1. Experimental procedures... 100
V.2. List of abbreviations... 104
V.3. References ... 105 V.4. Curriculum vitae ...Error! Bookmark not defined.
I. Summary
Teneurins are large transmembrane proteins playing fundamental roles in development.
They are highly expressed in the developing and adult nervous system in distinct layers, often in interconnected regions of the brain, where they were proposed to have an important function during target recognition and synapse formation. Beside the nervous system, teneurins are found at places of cell migration, at morphogentically active zones in developing limbs or at muscle attachment sites. The extracellular domains of teneurins interact in a homophilic manner and this interaction may trigger release of the intracellular domain from the membrane. The soluble intracellular domain can translocate to the nucleus and influence gene expression.
To elucidate teneurin function, we studied the role of the single teneurin orthologue ten-1 in C. elegans development. We characterized mutants in the ten-1 gene and found that TEN-1 is important for gonad development, vulva formation, distal tip cell migration and axonal guidance. Despite of such pleiotropic phenotypes, we initially concentrated on the gonadal defects. We found that ten-1 does not control germline proliferation but is essential for the maintenance of the gonadal basement membrane. The basement membrane defect in the ten-1 mutant was very local and most of the basement membranes showed generally wild-type ultrastructure as analyzed by electron microscopy. Similar disorganization of early gonads has been reported for integrin ina-1, dystroglycan dgn-1 and laminin epi-1 mutant worms. Therefore, we took a candidate gene approach and tested the genetic interactions between ten-1 and genes encoding various basement membrane proteins and receptors. This analysis revealed that teneurin acts redundantly with integrin and dystroglycan. Moreover, mutation in ten-1 sensitized the worms to loss of nidogen and led to defects in pharyngeal morphogenesis. Genetic studies also indicated that laminin could be a ligand for TEN-1 but initial data from vertebrate in vitro studies have not confirmed this hypothesis. Reporter constructs showed TEN-1 localization in the cytoplasm and membrane of certain head neurons, pharynx and several gonadal cells but no signs of nuclear translocation of the teneurin intracellular domain could be detected.
INTRODUCTION
II. Introduction
II.1. Teneurins
Teneurins are large transmembrane proteins with fundamental functions during development in regulating cell-cell interactions and cell adhesion (Tucker and Chiquet- Ehrismann, 2006; Tucker et al., 2007). They are phylogenetically conserved from Caenorhabditis elegans to man and were described in several species, including ten-m/odz and ten-a in Drosophila (Baumgartner et al., 1994; Fascetti and Baumgartner, 2002; Levine et al., 1994; Rakovitsky et al., 2007), ten-1 in C. elegans (Drabikowski et al., 2005), zebrafish (Mieda et al., 1999), chicken (Minet et al., 1999; Rubin et al., 2002;
Tucker et al., 2001; Tucker et al., 2000), rat (Otaki and Firestein, 1999), mouse (Ben-Zur et al., 2000; Oohashi et al., 1999; Zhou et al., 2003), and man (Minet and Chiquet- Ehrismann, 2000). In vertebrates, four teneurin paralogs exist and they were named teneurin-1 to -4, ten-m1 to -m4 or odz-1 to -4.
II.1.1. Protein domains and structure
Teneurins are type II transmembrane proteins with an approximate molecular mass of 300 kDa. They have an N-terminal cytoplasmic domain and a large extracellular part.
The extracellular domain of all teneurins is highly conserved and contains eight tenascin- type EGF-like repeats, a region of conserved cysteines and YD repeats. The second and fifth EGF-like repeat have an odd number of cysteines and it was proposed that the unpaired cysteines may form disulfide bridges with adjacent teneurin molecule leading to homo- or heterodimer formation (Fig. II.1A) (Feng et al., 2002; Oohashi et al., 1999).
The EGF-like repeats are followed by a region containing 17 cysteines that are conserved throughout family members in all species and may be required for correct protein folding.
Finally, the C-terminal half of the extracellular domain contains 26 YD repeats. This motif is only found in some bacterial proteins (e.g. rearrangement hot spot elements in E.
coli) and it is predicted to be highly glycosylated (Feng et al., 2002; Minet and Chiquet- Ehrismann, 2000).
A
B
C
Figure II.1. (A) Teneurins are type II transmemebrane proteins connected through covalent bonds in the second and fifth EGF-like repeat. Large globular domains are present in the C-terminal part of the molecule. (B) Domain organization of vertebrate teneurins. The intracellular domain contains nuclear localization signal (NLS), EF-hand like motifs (EF), proline-rich stretches (PP) and putative tyrosine phosphorylation sites (Y). The single transmembrane domain is followed by a large extracellular part consisting of eight tenascin-type EGF-like repeats, a region with conserved cysteines and YD repeats.
Three proteolytic cleavage sites are indicated by arrows. (C) Domain organization of C. elegans TEN-1.
TEN-1L contains proline-rich stretch, putative tyrosine phosphorylation sites and nuclear localization
INTRODUCTION
Intracellular domains of teneurins show little conservation between the phyla and cannot be aligned in a linear manner. However, most of them contain proline-rich stretches, putative tyrosine phosphorylation sites and nuclear localization signals. The domain organization and predicted structures of teneurins are shown in Fig. II.1A,B.
In Caenorhabditis elegans there is a single teneurin ortholog, named ten-1. This gene is under control of alternative promoters giving rise to two protein variants, differing in the length of their intracellular domain (Fig. II.1C). The overall domain organization of TEN-1 is highly similar to vertebrate teneurins and the TEN-1 long variant contains a proline-rich stretch and a putative bipartite nuclear localization signal in its cytoplasmic part (Drabikowski et al., 2005).
II.1.2. Teneurin expression patterns
The main site of teneurin expression is the developing and adult nervous system (Ben- Zur et al., 2000; Mieda et al., 1999; Oohashi et al., 1999; Otaki and Firestein, 1999;
Rubin et al., 2002; Tucker et al., 2000; Zhou et al., 2003). Teneurin paralogs are often found in subpopulations of neurons in the developing brain and their expression patterns are largely non-overlapping (Rubin et al., 2002; Rubin et al., 1999; Zhou et al., 2003).
For instance, teneurin-1 and -2 are mainly localized in interconnected regions of specific visual pathways, i.e. teneurin-1 is expressed in the tectofugal pathway, while teneuirn-2 is primarily found in the thalamofugal pathway. All four teneurins are also expressed in distinctive, complementary gradients in the developing mouse cortex as well as thalamic nuclei that are connected with appropriate cortical regions. Teneurins show reduced expression in the neocortex of Emx2-/- mice and it was proposed that they function in cortical arealization (Li et al., 2006). Moreover, in vitro and in vivo studies showed that teneurins promote neurite outgrowth implying their important role in axon guidance, target recognition and establishing neuronal connectivity (Leamey et al., 2007a; Minet et al., 1999; Rubin et al., 1999).
Similarly to vertebrate teneurins, ten genes in Drosophila are expressed in subsets of neurons (Baumgartner et al., 1994; Fascetti and Baumgartner, 2002; Levine et al., 1994)
lobes (Minet et al., 1999). Furthermore, expression of both C. elegans isoforms is found in a subpopulation of neurons (Drabikowski et al., 2005).
Besides the nervous system, teneurins are often expressed at sites of pattern formation or cell migration. Some avian teneurins were shown to be expressed at morphogenetically active sites of developing limbs, pharyngeal arches, somites and notochord (Tucker et al., 2001; Tucker et al., 2000). Drosophila ten-m is found in alternating stripes of the developing embryo, tracheal system, muscle attachment sites and cardiac cells (Baumgartner and Chiquet-Ehrismann, 1993; Baumgartner et al., 1994).
In C. elegans, expression of ten-1 from the upstream promoter is mainly detected in the cells of mesodermal origin, like somatic gonad cells (including distal tip cells), pharynx, some muscle and hypodermal cells. The downstream promoter is mainly active in the ectoderm, e.g. dorsal hypodermal cells and leader cells during morphogenesis, arcade cells and excretory duct during postembryonic development (Drabikowski et al., 2005).
II.1.3. Teneurin function – knockout studies
Fundamental roles of teneurins in development have been demonstrated by genetic studies, mostly in invertebrates. Drosophila ten-m mutants are embryonic lethal due to the fusion of adjacent denticle belts (Baumgartner et al., 1994; Levine et al., 1994).
Moreover, late ten-m mutants show defects in ventral nerve cord development, cardiac cells and eye patterning (Kinel-Tahan et al., 2007; Levine et al., 1994). Similar defects in cuticle formation and eye development have been described for the second Drosophila gene, ten-a (Rakovitsky et al., 2007). In Caenorhabiditis elegans, the single ten-1 gene is required for several aspects of cell migration and morphogenesis. Mutations in the ten-1 gene (or its knock down by RNAi) result in a pleiotropic phenotype, including ectopic germline formation, gonad disorganization, distal tip cell migration and axonal guidance defects as well as nerve cord defasciculation (Drabikowski et al., 2005).
Recently, the first vertebrate knockout has been described (Leamey et al., 2007b).
Mutation in the mouse teneurin-3 gene leads to defects in eye-specific patterning in the
INTRODUCTION
phenotype of teneurin-3 knockout mice may be the result of compensation by other family members.
II.1.4. Teneurin processing
Several reports postulate that teneurins undergo proteolytic processing. A putative furin cleavage site is located between the transmembrane domain and the EGF-like repeats (number 1 on Fig. II.1B). This site is present in all mouse teneurins, both Drosophila ten- m and ten-a, as well as C. elegans ten-1 (Drabikowski et al., 2005; Oohashi et al., 1999).
Processing at this cleavage site leads to the release of the extracellular domain from the cell surface. Immunostaining with the antibody against the extracellular domain of teneurin-2 co-localizes with the laminin staining in certain chicken basement membranes suggesting that the shed extracellular part may bind to the surrounding extracellular matrix (Tucker et al., 2001). It was also shown that teneurin-2 can be cleaved in vitro at the furin site and soluble ten-m can be found in the conditioned medium of Drosophila Schneider S2 cells (Baumgartner et al., 1994; Rubin et al., 1999).
Several lines of evidence indicate that teneurins undergo regulated intramembrane proteolysis and may be cleaved near or in the transmembrane domain (number 2 on Fig.
II.B). As a result, the intracellular domain is released from the membrane and can translocate to the nucleus. For teneurins, neither the exact cleavage site nor the protease(s) have been identified so far.
However, it was shown that there is a functional interaction between teneurin-2 and the zic-1 transcription factor (Bagutti et al., 2003). The intracellular domain of teneurin-2 could be detected in the nuclei of HT1080 cells in discrete spots that often co-localize with endogenous PML (promyelocytic leukemia protein). The translocation of the intracellular domain into the nucleus was confirmed in vivo for C. elegans TEN-1 (Drabikowski et al., 2005). An antibody against the intracellular domain stains both the membrane and nuclei of developing embryos, in contrast to the antibody against the C- terminal part of TEN-1 that labels membranes exclusively.
Finally, there are some reports indicating that teneurins may be processed at a furin site
associated peptide (TCAP) shows homology to corticotrophin releasing factor family and may modify neurite outgrowth in immortalized hypothalamic cells (Al Chawaf et al., 2007; Wang et al., 2005).
II.1.5. Teneurin interacting proteins
To uncover the biological function of teneurins, several efforts have been made to identify teneurin interacting proteins. Two such proteins have been found in a yeast two- hybrid screen using part of the teneurin-1 intracellular domain as bait (Nunes et al., 2005). One of them is CAP/ponsin, a cytoskeleton adapter protein playing an important role in cell adhesion (Zhang et al., 2006). Another one, MBD1 (a methyl CpG binding protein) is a known transcriptional repressor (Wade, 2001). The interaction between teneurin-1 and these two proteins was confirmed by immunoprecipitation and co- localization studies. The biological function of teneurin-1 binding to CAP/ponsin or MBD1 is unclear but it may be required for a connection to the actin cytoskeleton or the transcriptional regulation.
Teneurins are also thought to interact in a homophilic manner in their extracellular domains (Leamey et al., 2007a; Rubin et al., 2002) and most likely with other cell-surface or extracellular ligands but till to date, none of them has been identified. Our working model predicts that homophilic interaction or ligand binding initiates cytoskeletal changes and/or proteolytic release of the teneurin intracellular domain and its translocation to the nucleus (Fig. II.2).
INTRODUCTION
Figure II.2. Model of teneurin signaling. Ligand binding or homophilic interaction between teneurin extracellular domains triggers cytoskeletal remodeling or proteolytic release of the intracellular domain.
The soluble intracellular domain can translocate to the nucleus and associate with PML bodies, or bind to nuclear proteins (e.g. Zic or MBD1) and regulate gene expression. Picture is taken from (Kenzelmann et al., 2007).
II.2. Caenorhabditis elegans as a model system
In this study, we took advantage of C. elegans as our model system. This soil nematode has been widely used in research because of its simple anatomy, short life cycle, invariant cell lineage, powerful genetics and simplicity of culturing conditions. Worms provide an excellent in vivo model to study a variety of processes, e.g. cell migration (distal tip cells), cell invasion (anchor cell) or mechanisms of morphogenesis (epidermis). Forward genetic screens allow identifying the C. elegans mutations that produce certain phenotypes and have led to the discovery of key proteins required for fundamental
II.2.1. Basement membranes and their receptors
Basement membranes (BMs) are thin, specialized sheets of extracellular matrix proteins that separate tissues and organs, and are required for cell adhesion, migration and differentiation during development (Schwarzbauer, 1999; Yurchenco et al., 2004). Many basement membrane proteins and receptors found in vertebrates are conserved in C.
elegans (Cox et al., 2004; Hutter et al., 2000) but there are less genes and isoforms in each family. The major BM molecules and receptors described in worms are shown in Figure II.3.
collagen IV emb-9, let-2
nidogen nid-1
perlecan unc-52 laminin
epi-1, lam-3, lam-1, lam-2
syndecan
sdn-1 integrins
ina-1, pat-2, pat-3 dystroglycan
dgn-1
SPARC ost-1 collagen XVIII
cle-1
Figure II.3. Basement membrane proteins and receptors. Gene names are in italic. Adapted from (Kramer, 2005; Yurchenco et al., 2004).
The composition of basement membranes differs between tissues and developmental stages, e.g. collagen IV EMB-9/LET-2 and nidogen NID-1 are widely present in worm basement membranes, while perlecan UNC-52 is restricted to basement membranes surrounding muscle cells and a LET-2 (exon 9) splice variant predominates during embryogenesis (Graham et al., 1997; Kang and Kramer, 2000; Mullen et al., 1999; Sibley et al., 1993). Basement membranes in worms have an asymmetric appearance between
INTRODUCTION
electron micrographs (Fig. II.4E,F) (Huang et al., 2003). All basement membranes in C.
elegans seem to consist of a single sheet although the epidermal basement membranes have often a “lollypop” appearance (dark dots sticking out of BM), which may represent an additional layer.
Figure II.4. Basement membrane ultrastructure in C. elegans. (A) A thin basement membrane (arrows) is present on the gonadal sheath (s), while there is no BM (arrowheads) separating sheath cell and germ cells (g). (B) A basement membrane (arrowhead) starts to form around the developing oocyte (o). (C) Two mature oocytes (o) are ensheathed by BMs (arrowheads). (D) Male tail cross section. A thick BM (arrowhead) covers cloacal muscles (c) on the cuticle side. (E) Body wall muscles are covered by a thick BM (arrowheads) on the side of epidermis and a thin BM (arrows) on the pseudocoelomic side. (F) The pharynx (ph) is ensheathed by a thick BM (arrowheads) (reproduced from Huang et al., 2003).
Laminins
Laminins are secreted heterotrimeric molecules that are able to self-polymerize and form networks (Miner and Yurchenco, 2004). The C. elegans genome encodes two laminin α chains: LAM-3 and EPI-1, a single β chain LAM-1 and a single γ chain LAM-2. LAM-3 αA and EPI-1 αB chains show high similarity to vertebrate α1/α2 and α3/α4/α5 chains, respectively (Hutter et al., 2000). Both laminin isoforms are broadly distributed among worm basement membranes but EPI-1 is associated with epidermal and gonadal BMs exclusively, and LAM-3 is unique for the nervous system (Huang et al., 2003).
C. elegans laminins are required for cell polarity, differentiation, migration and tissue separation. Mutations in the lam-3 gene causes complete developmental arrest during embryogenesis or at the L1 stage due to pharynx deformation. In lam-3(n2561) arrested larvae, pharyngeal cells adhere to their surrounding tissues, while most of the other tissues and organs seem to be normal (Huang et al., 2003). The majority of epi-1 null mutants arrest as embryos or early larvae, however 27% of worms develop to adulthood.
Adult epi-1 mutants show disruption of basement membranes, muscle polarization defects, axon misguidance and germ cell invasion into adjacent tissue due to gonad epithelialization failure (Huang et al., 2003).
Reduction of lam-1 or lam-2 (or both lam-3 and epi-1) function by RNAi result in high embryonic lethality (80-85%) due to cell detachment and severe disorganization of developing embryos. Furthermore, partial loss-of-function mutants in the lam-1 gene show similar phenotypes to viable laminin α mutants, implying that both α and β subunits are required for basement membrane assembly and integrity (Kao et al., 2006).
Nidogen
The single C. elegans nidogen NID-1 is broadly distributed among BM but is particularly concentrated around the developing gonad and nervous system. There are three nid-1 splice variants, which show differential expression during development (Kang and Kramer, 2000). The cg119 null mutant is viable and fertile but shows defects in synapse organization and function (Ackley et al., 2005; Ackley et al., 2003). However, this
INTRODUCTION
loss-of-function mutation causes defects in dorso-ventral positioning of specific axons indicating that nidogen plays an important role in axon sorting along the midline (Kim and Wadsworth, 2000).
Perlecan
Perlecan is a major heparan sulphate proteoglycan of basement membranes and in worms it is encoded by a single unc-52 gene. Three main isoforms are expressed from the unc-52 locus: short (S), medium (M) and large (L) (Mullen et al., 1999). Additionally, several alternative splicing sites exist, therefore as many as 50 perlecan isoforms may be present in C. elegans (Rogalski et al., 2001). UNC-52 localization is limited to muscle cells. In body wall muscles, perlecan is found in the basement membrane between muscles and epidermis and is concentrated at the dense bodies and M-lines (Mullen et al., 1999).
Several mutant alleles of the unc-52 gene have been isolated and their analysis revealed that the M isoform is essential for myofilament assembly. Null mutants of the unc-52 gene arrest at the two-fold stage because of myofilament lattice disorganization, while mutations eliminating only the L isoform show essentially wild-type phenotype (Mullen et al., 1999). Loss-of-function mutation, e444, leads to progressive paralysis and gonad disorganization in adults indicating that it affects only some perlecan isoforms specific for adult worms (Gilchrist and Moerman, 1992).
Collagen XVIII
The C. elegans gene cle-1 is a single ortholog of vertebrate collagens type XV/XVIII.
Expression from three different promoters gives rise to three protein isoforms (A-C), which share a common endostatin domain. CLE-1 is broadly distributed in BMs but is concentrated in the nervous system. Loss-of-function mutation in cle-1 results in neuron and distal tip cell migration defects, male tail defects and low penetrance larval arrest (Ackley et al., 2001). It was also shown that CLE-1 is required for synapse organization and function (Ackley et al., 2003). Moreover, it may stabilize the basement membranes as cle-1 overexpression rescues gonad fragility defects found in fibulin fbl-1 mutants (Muriel et al., 2006).
Integrins
Integrins are heterodimeric ECM receptors consisting of one α and one β subunit. The C.
elegans genome encodes two α chains: INA-1 and PAT-2, and a single β chain, PAT-3.
INA-1/PAT-3 is mostly similar to laminin-binding integrins, while PAT-2/PAT-3 shows high similarity to RGD-binding integrins (Bokel and Brown, 2002).
INA-1 is broadly expressed in developing embryos. However, in L1 larvae it becomes restricted to migrating cells (including distal tip cells) and neurons, as well as organs undergoing morphogenesis (e.g. vulva and uterus). Null mutation of the ina-1 gene leads to developmental arrest at the L1 stage due to pharyngeal malformation. Weak loss-of- function ina-1 mutants are viable but show defects in the anterior hypoderm (notched head phenotype), axon defasciculation, and disorganization of the developing gonad (Baum and Garriga, 1997).
Both PAT-2 and PAT-3 integrin chains are strongly expressed in muscle cells (Gettner et al., 1995; Williams and Waterston, 1994). Mutations in pat-2 or pat-3 genes cause the Pat (paralyzed at two-fold) phenotype – mutant embryos fail to complete morphogenesis and arrest at two-fold stage due to sarcomere disorganization (Williams and Waterston, 1994). This phenotype is similar to defects observed in lethal unc-52 mutants (Rogalski et al., 1993) implicating that PAT-2/PAT-3 integrin may be an essential receptor required for perlecan binding in muscle cells.
The role of PAT-3 integrin in larval and adult tissues was investigated by a dominant negative approach. Expression of a HA-βtail transgene (HA-tagged transmembrane and cytoplasmic domain) in gonad, body wall and sex muscles leads to uncoordinated, egg- laying phenotype and gonad migration defects (Lee et al., 2001).
Dystroglycan
In vertebrates a single dystroglycan exists, while the C. elegans genome contains three dystroglycan related genes: dgn-1, dgn-2 and dgn-3. C. elegans dgn-1 shows the highest similarity to vertebrate and Drosophila dystroglycans, although the protein is not processed into α and β subunits. DGN-1 is highly expressed in epithelial cells (including
INTRODUCTION
development (Johnson et al., 2006). In vertebrates, dystroglycan hypoglycosylation in skeletal muscles leads to muscular dystrophies (Haliloglu and Topaloglu, 2004). In contrast, worm DGN-1 is not expressed in muscles and it does not interact genetically with genes encoding components of the dystrophin complex (Johnson et al., 2006).
Mutations in dgn-2 or dgn-3 do not cause any obvious defects and their expression patterns have not been described in detail, therefore their function remains unknown (James M. Kramer, unpublished).
II.2.2. Epithelial morphogenesis
In C. elegans, epithelial cells play a crucial role in the process of embryonic morphogenesis as they determine the shape of the embryo. Epidermal morphogenesis requires changes in the position and shape of epidermal cells as well as their interaction with underlying neuronal cells and body wall muscles. Once the epidermal cells are specified, three major steps of morphogenesis take place: movements of ventral neuroblasts, ventral enclosure and embryo elongation (developmental stages and timing are shown in Fig. II.5).
In the first step of epidermal morphogenesis, movements of ventral neuroblasts are required for closure of the gastrulation cleft. Several signaling pathways were shown to be essential for this process, including ephrin (VAB-1, EFN-1 to -4) and semaphorin-2A (MAB-20) pathway components, and the LAR receptor protein tyrosin phosphatase (PTP-3) (Chin-Sang et al., 1999; Chin-Sang et al., 2002; Harrington et al., 2002; Roy et al., 2000; Wang et al., 1999). Mutants in these receptors or ligands show an enlarged and persistent ventral cleft. Disorganization of ventral neuroblasts, which serve as a substrate for epidermal cells, affects the subsequent step of morphogenesis, epidermal enclosure.
Before the ventral enclosure begins, dorsal epidermal cells rearrange to form a single row of cells (process known as dorsal intercalation). Afterward in the process of ventral enclosure, epidermal cells from the dorsal side of the embryo migrate ventrally to close up at the ventral midline (Williams-Masson et al., 1997). In the first step, two pairs of leading cells extend their long protrusions towards the ventral midline and rapidly form
junctions between their counterparts. Subsequently, posterior ventral cells fill the ventral pocket and enclose by a purse-string mechanism.
Figure II.5. Epidermal morphogenesis timing and developmental stages. Times are indicated for embryonic development at 20°C. Nomarski pictures show worm development at respective stages. Picture is taken from (Chisholm and Hardin, 2005).
Modulation of the actin cytoskeleton is essential for the process of ventral enclosure.
Mutants in Rac GTPase, ced-10, and its interacting proteins, gex-2 and gex-3, as well as the Arp2/3 complex required for microfilament nucleation, show disorganized epidermis (Severson et al., 2002; Soto et al., 2002). Moreover, components of the cadherin/catenin complex, i.e. HMR-1/cadherin, HMP-1/α-catenin and HMP-2/β-catenin, are required for
INTRODUCTION
In the last step of morphogenesis, elongation, the embryo reduces its diameter and epidermal cells elongate along the anterior-posterior axis. Similarly to ventral enclosure, both reorganization of the actin cytoskeleton and the cadherin/catenin complex are required for the early steps of the elongation process (Costa et al., 1998; Wissmann et al., 1997).
Interaction between epidermal cells and underlying body wall muscles appears to be critical for later stages of elongation as mutations completely eliminating the muscle function cause developmental arrest at the two-fold stage (Williams and Waterston, 1994). Several basement membrane components playing an important role in muscle development are essential for the elongation process. Perlecan/UNC-52 is required for myofilament lattice assembly and collagen IV EMB-9/LET-2 - to maintain the muscle- epidermis attachment during muscle contraction (Gupta et al., 1997; Hresko et al., 1994).
Mutants in all these genes arrest during elongation.
In addition, components of fibrous organelles, which transmit the forces of muscle contraction to epidermis and cuticle, are required for the elongation process. They include myotactin/LET-805, spectraplakin/VAB-10 and intermediate filament proteins IFA-3, IFB-1 (Bosher et al., 2003; Hresko et al., 1999; Woo et al., 2004).
II.2.3. Pharynx development
The pharynx is a linear tube with two bulbs and is ensheathed by a thick basement membrane. It can be divided into six parts: the buccal cavity, procorpus, anterior bulb (metacorpus), isthmus, terminal bulb and pharyngeal-intestinal valve (Fig. II.6). There are seven cell types that form the pharynx: arcade cells, muscles, epithelia, neurons, glands, mariginal cells and valves (Fig. II.6) (Albertson and Thomson, 1976).
Figure II.6. Pharynx organization. Nuclei of different cell types are labeled as follows: red - muscles, purple – neurons, orange – epithelia, pink – mariginal cells, brown – glands. Picture is taken from (Mango, 2007).
The pharyngeal cells are derived from ABa and MS descendants (Sulston et al., 1983) and pharynx development is predominantly regulated by the transcription factor PHA-4 (Gaudet and Mango, 2002; Mango et al., 1994). At the end of gastrulation, the pharyngeal primordium forms an epithelialized ball of cells connected by adherens junctions (Portereiko and Mango, 2001). Subsequently, the foregut connects to the buccal cavity in the morphogenetic process called pharyngeal extension, which can be divided into three steps: (I) rotation of pharyngeal cells, (II) epithelialization of arcade cells and (III) contraction (Fig. II.7) (Portereiko and Mango, 2001).
INTRODUCTION
Figure II.7. Three steps of pharyngeal extension. Cell membranes are labeled in red with anti-β- spectrin/UNC-70 antibody and adherens junctions are green. Basement membranes are represented by dotted yellow line. Picture is taken from (Mango, 2007).
In the first stage, pharyngeal cells reorient their apical and basolateral polarity relative to the embryonic axes and align with the arcade cells. The basement membrane at the anterior tip of the primordium is removed and pharyngeal cells at this position lose cell contacts. Subsequently, arcade cells (mesenchymal) are converted into epithelial cells and form adherens junctions connecting them to the pharyngeal epidermis. Finally, pharyngeal and arcade cells undergo local contraction that pulls them tightly together.
Mutations in several genes affect pharyngeal morphogenesis causing a Pun (pharynx unattached) phenotype. However, it is unclear, whether they are required for correct cell fate determination, cell differentiation or morphogenesis. Proper pharyngeal attachment requires several transcription factors, e.g. ast-1 (Schmid et al., 2006), die-1 (Heid et al.,
conjugating enzyme ubc-18 (Fay et al., 2003) or ubiquitin-ligase ari-1 (Qiu and Fay, 2006).
Interestingly, many proteins required for the formation or maintenance of epithelia, like cadherins, catenins or discs-large, are not essential for pharyngeal morphogenesis (Costa et al., 1998; Firestein and Rongo, 2001; Pettitt et al., 1996). However, mutation in zen-4 or cyk-4 cause a Pun phenotype since the kinesin-like protein ZEN-4 and RhoGAP CYK- 4 were shown to be important for arcade cell polarization (Portereiko et al., 2004).
II.2.4. Gonad development
The gonad primordium consists of four cells: Z1 and Z4 are somatic gonad precursor cells, whereas Z2 and Z3 are germline founder cells. Z1 and Z4 come from the MS lineage, are born late during embryogenesis and migrate to associate with germline precursor cells. Z2 and Z3 are exclusive descendants of P4 and during embryogenesis they are attached to the intestine (Sulston et al., 1983). At hatching, these four cells form a compact primordium which is completely ensheathed by a basement membrane.
Germline and somatic gonad precursor cells start to proliferate at the L1 stage. In late L2, somatic gonad cells reorganize and form the somatic gonad primordium of hermaphrodite (SPh) which separates proliferating germ cells into a posterior and an anterior population.
Distal tip cells remain at the tips of gonad arms controlling gonad migration and promoting germline mitosis. During L3 stage, proximal germ cells enter meiosis, while distal germline nuclei continue to divide mitotically. Gametogenesis starts at the proximal end of the gonad at the L4 stage and continues throughout adulthood (Hubbard and Greenstein, 2000). Gonad development is summarized in Fig. II.8.
Z1 and Z4 cells give rise to all somatic structures in the gonad, including distal tip cells, sheath cells, spermatheca and uterus (Kimble and Hirsh, 1979). Until late L2, 12 somatic cells are formed from Z1 and Z4 precursors: two distal tip cells, four sheath/spermatheca precursors, two dorsal and three ventral uterine cells, and a single anchor cell (McCarter et al., 1997). Already at these early steps of gonad development, descendants of Z1/Z4
INTRODUCTION
Sheath cells play an important role in the maintenance of gonadal integrity and gametogenesis since they provide nutritional and structural support, and control meiotic progression (Hall et al., 1999; McCarter et al., 1997). The sheath/spermathecal (SS) precursor cells and their descendants are required for germline proliferation (Killian and Hubbard, 2005; McCarter et al., 1997). Moreover, proximal sheath cells and spermatheca play an important role in ovulation (McCarter et al., 1999).
Figure II.8. Gonad development in C. elegans. The developing gonad is shown at the L1 stage, L1/L2 molt, L3 stage, L3/L4 molt and adult stage. Germ cells (GC) are shown in blue, distal tip cells (DTC) are red, sheath/spermatheca precursors (SS) are yellow, uterine cells are green (ventral uterine - VU, dorsal uterine – DU), and anchor cell (AC) is pink. Cells forming somatic gonad primordium (SPh) are underlined. In adult worms, both germline and somatic structures (including five pairs of sheath cells) are shown. Picture is taken from (Hubbard and Greenstein, 2000).
One of the somatic cells, the anchor cell (AC), plays a crucial role in vulva induction and the formation of the connection between uterus and vulva. During the L3 stage, the AC attaches to the ventral side of the gonadal basement membrane, removes the gonadal and epidermal BMs precisely at its basolateral side and invades the underlying tissue (Fig.
II.5). The anchor cell invasion is studied as an in vivo model of regulated invasion. The transcription factor FOS-1 is a key regulator of this process and it affects the transcrption of three targets: ZMP-1/matrix metalloprotease, CDH-3/protocadherin and ECM protein hemicentin (Sherwood, 2006).
Figure II.5. Anchor cell (AC) invasion through the basement membranes. Anchor cell is labeled by GFP expressed under cdh-3 promoter. In the early L3 stage (a), AC attaches to the ventral side of the gonadal basement membrane, just above P6.p vulva precursor cell. Gonadal and epidermal BMs are removed during the mid L3 stage (b). During mid-to-late L3 stage (d), AC is invading between vulval cells. By the early L4 stage (d), AC invasion is complete. Immunostaining with laminin antibody reveals loss of BMs precisely under the AC (e). Reproduced from (Sherwood, 2006).
INTRODUCTION
II.3. Aim of the work
Since little is known about teneurin function in vivo, we took advantage of the C. elegans model organism to elucidate the role of the single teneurin gene ten-1 during worm development. As ten-1 mutant worms showed a pleiotropic phenotype with many tissues affected, we initially concentrated on gonadal defects. We discovered that TEN-1 is essential for the maintenance of the gonadal BM during development by analyzing BM organization with GFP markers and at high resolution by transmission electron microscopy. In addition, we used a candidate gene approach to identify receptors and pathways acting redundantly to ten-1 and found several synergistic genetic interactions between ten-1 and mutants in BM components and receptors. To shed light on the mechanism of teneurin action in preserving basement membrane integrity, we characterized the defects found in synthetic lethal double mutants and investigated TEN-1 localization.
III. Results
III.1. Results – published
III.1.1. Teneurins – proteins with fundamental roles in development
Richard P. Tucker, Daniela Kenzelmann, Agnieszka Trzebiatowska, and Ruth Chiquet- Ehrismann
Int J Biochem Cell Biol, 2007, 39: 292-297
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
III.1.2. ten-1, an essential gene for germ cell development, epidermal morphogenesis, gonad migration, and neuronal pathfinding in Caenorhabditis elegans
Krzysztof Drabikowski, Agnieszka Trzebiatowska, and Ruth Chiquet-Ehrismann
Dev Biol, 2005, 282: 27-38
My contribution: I characterized ten-1(ok641) worms and analyzed the phenotypes in the mutant and rescued worms.
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
RESULTS - PUBLISHED
III.2. Results – submitted
III.2.1. C. elegans teneurin, ten-1, is required for gonadal and pharyngeal basement membrane integrity and acts redundantly with integrin ina-1 and dystroglycan dgn-1
Agnieszka Trzebiatowska, Krzysztof Drabikowski, Ulrike Topf, and Ruth Chiquet- Ehrismann*
Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland
Short title: TEN-1 function in basement membranes
Abbreviations: BM, basement membrane; DIC, differential interference contrast; L1, first larval stage; L2, second larval stage; L3, third larval stage; L4, fourth larval stage; Pun, Pharynx Unattached; SGP, somatic gonad precursor cells.
*Corresponding author: Ruth Chiquet-Ehrismann
Friedrich Miescher Institute for Biomedical Research Novartis Research Foundation
Maulbeerstrasse 66 CH-4058 Basel Switzerland
Tel. + 41 61 697 24 94
RESULTS - SUBMITTED
Abstract
The C. elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. ten-1 null mutants are sterile due to local basement membrane deficiency leading to early gonad disruption. They also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with ina-1 and dgn-1 indicating that TEN-1 could be a receptor acting redundantly with integrin INA-1 and dystroglycan DGN-1. The morphological defects found in epi-1 deficient worms are enhanced by lack of ten-1 suggesting that laminin EPI-1 is a potential extracellular ligand for TEN-1. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. TEN-1 appears to be an important receptor required for basement membrane maintenance and/or adhesion in particular organs and cells.
Introduction
Teneurins are large transmembrane proteins that play important roles in cell signaling and cell adhesion (Tucker and Chiquet-Ehrismann, 2006; Tucker et al., 2007). Teneurins are phylogenetically conserved among metazoans and they were described in several species, including ten-1 in Caenorhabditis elegans (Drabikowski et al., 2005), ten-m/odz and ten- a in Drosophila (Baumgartner et al., 1994; Fascetti and Baumgartner, 2002; Levine et al., 1994; Rakovitsky et al., 2007), zebrafish (Mieda et al., 1999), chicken (Minet et al., 1999; Rubin et al., 2002; Tucker et al., 2001; Tucker et al., 2000) and mouse (Ben-Zur et al., 2000; Oohashi et al., 1999; Zhou et al., 2003). In vertebrates, four teneurin paralogs exist and they were named teneurin-1 to -4, ten-m1 to -m4 or odz-1 to -4.
The extracellular domain of all teneurins is composed of eight tenascin-type EGF-like repeats, a region of conserved cysteines, and YD repeats which are found in a few bacterial proteins (Minet and Chiquet-Ehrismann, 2000). The intracellular domain contains proline-rich stretches and putative tyrosine phosphorylation sites but is less conserved than the extracellular part and cannot be aligned in a linear way between the phyla. Teneurins are thought to interact in a homophilic manner (Bagutti et al., 2003;
Leamey et al., 2007a; Oohashi et al., 1999; Rubin et al., 2002) and to date, no other ligand has been identified.
The name “teneurins” refers to their high expression in the developing and adult nervous system (Ben-Zur et al., 2000; Mieda et al., 1999; Oohashi et al., 1999; Otaki and Firestein, 1999; Rubin et al., 2002; Tucker et al., 2000; Zhou et al., 2003). In the developing mouse cortex, all teneurins are expressed in distinctive gradients and may be required for neocortical patterning (Li et al., 2006). Several reports point out their role in the development of visual pathways. Leamey and co-workers (2007a) have found that teneurins are upregulated in visual versus somatosensory areas of the neocortex.
Moreover, expression of different teneurins is largely non-overlapping and can be found in interconnected regions of the developing visual system (Leamey et al., 2007a; Rubin et al., 2002; Rubin et al., 1999). For instance, teneurin-1 staining is found in the tectofugal pathway and teneurin-2 is primarily expressed in the thalamofugal pathway. In addition,
RESULTS - SUBMITTED
teneurins in axon guidance and target recognition. Recently, the first vertebrate teneurin knockout was described (Leamey et al., 2007b). Teneurin-3 regulates eye-specific patterning in the visual system and the knockout mice show impaired binocular vision.
Such a mild phenotype may be a result of functional redundancy and compensation by other teneurins.
Beside prominent expression in the nervous system, teneurins are also found in non- neuronal tissues. They are expressed in alternating stripes of the fly embryo, cardiac cells, muscle attachment sites, tracheal system in Drosophila (Baumgartner and Chiquet- Ehrismann, 1993; Baumgartner et al., 1994), limb buds, branchial archs, somites in chicken (Tucker et al., 2001; Tucker et al., 2000) or gonadal somatic cells, pharynx and muscles in Caenorhabditis elegans (Drabikowski et al., 2005). Teneurin expression in these tissues is often associated with pattern formation and cell migration.
The in vivo function of teneurins is mainly inferred from studies of Caenorhabdities elegans and Drosophila mutants. Mutation in the fly ten-m gene causes embryonic lethality due to the fusion of adjacent denticle belts (Baumgartner et al., 1994; Levine et al., 1994). Moreover, defects in the ventral nerve cord, cardiac cells and eye patterning are found in late ten-m mutant embryos (Kinel-Tahan et al., 2007; Levine et al., 1994).
Similar defects in cuticle and eye development have been observed for the second Drosophila teneurin gene, ten-a (Rakovitsky et al., 2007). In Caenorhabditis elegans, deletion in the ten-1 gene causes a pleiotropic phenotype, including ectopic germline formation, nerve cord defasciculation, defects in distal tip cell migration and axonal pathfinding (Drabikowski et al., 2005).
The single teneurin ortholog in Caenorhabditis elegans, ten-1, is under control of alternative promoters giving rise to two protein variants. The isoforms differ only in their intracellular domains and their expression patterns are complex but mostly non- overlapping: TEN-1 long (TEN-1L) is found mainly in the mesoderm, including pharynx, somatic gonad, various muscles and neurons, and TEN-1 short (TEN-1S) is predominantly expressed in some hypodermal cells and in a subset of neurons (Drabikowski et al., 2005).
We report here the role of TEN-1 in gonadal basement membrane maintenance and
Similar disorganization of the early gonads has been reported for basement membrane mutants, i.e. integrin α ina-1, dystroglycan dgn-1 and laminin αB epi-1 (Baum and Garriga, 1997; Huang et al., 2003; Johnson et al., 2006). Furthermore, the genetic interactions between ten-1, ina-1, dgn-1, epi-1 and nid-1 suggest that teneurin, integrin and dystroglycan have related and partly redundant functions in Caenorhabditis elegans development. We hypothesize that TEN-1 is a novel basement membrane receptor or regulator acting together with INA-1 and DGN-1.
RESULTS - SUBMITTED
Materials and Methods
General methods and C. elegans strains
C. elegans strains were maintained at 20˚C as described (Brenner, 1974). The following strains were used in this study: wild-type N2, variety Bristol, CH120: cle-1(cg120) I, CB444: unc-52(e444) II, VC518: ten-1(ok641) III; TM0651: ten-1(tm651) III; NG39:
ina-1(gm39) III; NG144: ina-1(gm144) III; CB189: unc-32(e189) III; CX2914:
nDf16/dpy-17(e164) unc-32(e189) III; CH119: nid-1(cg119) V; CH121: dgn- 1(cg121)/dpy-6(e14) unc-115(mn481) X. The tm651 deletion removes nucleotides R13F6: 3661-4550 of the ten-1 coding sequence.
The following GFP marker strains were used: RU7: kdEx7 [ten-1a::gfp]; RU97: ten- 1(ok641) kdEx45 [F36A3, III]; SS0747: bnIs1 [pie-1::GFP::PGL-1] (gift of Susan Strome); IM253: urEx131 [lam-1::gfp] (gift of William Wadsworth), CH1878: dgn- 2(ok209) dgn-3(tm1092) dgn-1(cg121); cgEx308 [DGN-1::GFP] (gift of James Kramer).
Double mutant worms were maintained as [ten-1(ok641);ina-1(gm144); kdEx45], [ten- 1(ok641/+);nid-1(cg119)], [ten-1(ok641);dgn-1(cg121/+); kdEx45] or [ten- 1(ok641/+);dgn-1(cg121); cgEx308] strains and genotyped by PCR for the phenotypic analysis.
Constructs and plasmids
The translational Pten-1a::GFP::TEN-1L minigene reporter construct was generated by cloning SpeI-HindIII cDNA fragment and HindIII-XhoI genomic fragment of TEN-1 long variant into p123T vector (Mo Bi Tec). The following restriction sites were introduced into the primers: SpeI and XhoI flanking the ten-1 coding sequence, SacII at the 5’ end of the ten-1a promoter and ApaI downstream of the 3’ UTR.
Long intracellular domain, transmembrane domain and a short fragment of the extracellular part were amplified using 5’-AACAGTCTACCGAATCCCAACC-3’ and 5’-ATAACTAGTATGTTCCAGCACAGGTAAACTACCACG-3’ primers and cDNA from mixed stage N2 worms as a template. For the extracellular domain of ten-1 we used
5’-GCTGAAATACCCACTCGCCAGC-3’ and 5’-
ATCTCGAGCTATTCAGATTTTCGGAACTTCC-3’ primers and R06H12 cosmid as a
site was mutated to CCTTGG. GFP was fused by PCR to the N-terminus of ten-1 cDNA fragment, which was cloned into SpeI-NcoI sites of ten-1 minigene. HA tag was added at the C-terminus of ten-1 coding sequence by PCR and cloned into HpaI-XhoI sites. The Pten-1a::GFP::TEN-1L construct contained 4235 bp of the ten-1a promoter and 512 bp sequence downstream of the stop codon. PCR fragments were generated with Pfu Turbo DNA polymerase (Stratagene).
Transgenic animals
Transgenic lines were generated as previously described (Mello et al., 1991). The Pten- 1a::GFP::TEN-1L plasmid was injected into ten-1(ok641) mutant worms. Injections of GFP::TEN-1 minigene at low concentration (5 ng/µl) resulted in a very weak GFP fluorescence, mainly in the nervous system. Therefore, we injected the worms with high concentrations of the transgene (40 ng/µl) and obtained several lines giving stronger GFP fluorescence. We used pRF4 [rol-6] as a co-injection marker.
RNA interference
RNA-mediated interference (RNAi) was performed as described (Kamath and Ahringer, 2003). The K08C7.3 RNAi clone was obtained from the Ahringer feeding library. Wild type and ten-1(ok641) synchronized L4 hermaphrodites were placed on RNAi plates and grown at 15˚C for 72 hours. Single adult worms were placed on fresh RNAi plates and allowed to lay eggs for 24 hours. These plates were examined for 3 days to determine embryonic lethality and postembryonic phenotypes.
Phenotypic analysis
Young adult hermaphrodites were placed on separate plates and allowed to lay eggs for 24 hours. The progeny was analyzed for embryonic and postembryonic phenotypes:
lethality, larval arrest, sterility and bursting at the vulva.
Time course of germline development
RESULTS - SUBMITTED
scored the number of germ cells in 20 worms for each genotype at following time points:
0, 8, 12, 16, 20 hours.
Microscopy
Animals were mounted on 2% agarose pads in a drop of M9 buffer containing 25 mM sodium azide. DIC and fluorescence images were acquired with Z1 microscope (Zeiss) and AxioCam Mrm camera (Zeiss) using 63x/1.4 NA Plan-APOCHROMAT objective (Zeiss) and AxioVision software.
Results
Both ten-1(ok641) and ten-1(tm651) are functional null alleles
In our previous study we described the ten-1 mutation, ok641, that carries an in-frame 2130 bp deletion removing four EGF-like repeats and a large part of the conserved cysteines region (Drabikowski et al., 2005). We now obtained another allele, tm651, lacking 890 bp and introducing a frameshift into the ten-1 coding sequence (Fig. 1). This deletion results in a loss of the transmembrane domain and the entire extracellular part.
Therefore, tm651 is most likely a null allele. Since phenotypes of both ten-1 mutants show similar penetrance (Table 1), we assume that ok641 represents a functional null allele as well.
To confirm this hypothesis, we created heterozygous worms carrying nDf16 deficiency in trans to tm651 or ok641 and investigated whether the mutant phenotypes became aggravated after complete removal of one copy of the ten-1 gene. The ok641/nDf16 and tm651/nDf16 worms displayed a similar range of defects to ok641 and tm651 homozygous animals and the values observed were very close to those calculated under the assumption of ten-1 mutants being null alleles (Table 2).
These data and the fact that ok641 and tm651 deletions affected protein regions that are common to both TEN-1 isoforms, suggested that there was no functional TEN-1 present in any of the ten-1 mutants.
Gonads of ten-1 mutant worms burst early in development
Previous studies demonstrated that TEN-1 plays an important role in gonad development and function (Drabikowski et al., 2005). Homozygous ten-1(ok641) worms are viable but 15-20% of them are sterile or burst through the vulva due to ectopic germline forming in the midbody region. Occasionally, gonads disintegrate completely and germ cells float in the pseudocoelom.
To determine the basis and the developmental stage of ectopic germline formation, we performed a time course experiment of germ cell proliferation in the early gonads of ten- 1(ok641) mutants. We used worms carrying a P-granule GFP marker to distinguish
RESULTS - SUBMITTED
2B). At the same time point, there were no germ cells present around the developing somatic gonad primordium in the wild type worms (Fig. 2A). A sharp DIC boundary surrounding the gonad was visible in the wild-type as well as a large part of ten-1(ok641) gonad (Fig. 2A,B) but absent on the dorsal side of the mutant gonad, where the germ cells leaked out into the pseudocoelom. Gonad bursting was not the result of germline overproliferation causing increased pressure on the gonadal basement membrane, since we did not find any difference in the number of germ cells between wild type and ten-1 mutants at this stage (Fig. 2C).
Gonadal basement membrane is not maintained in the ten-1 mutant
Bursting of the early gonads in the ten-1 mutant suggested that mutant worms have defects in the basement membrane formation or maintenance. Therefore, we examined the organization of the basement membrane in the ten-1(ok641) worms using a laminin-β LAM-1::GFP marker that labels all basement membranes in worms.
At hatching, both wild-type and ten-1 mutant gonad primordia were compact and completely surrounded by laminin (unpublished data). As the gonadal precursor cells divided, a discontinuity appeared in the ten-1(ok641) gonadal basement membrane. The laminin layer surrounding the developing gonad appeared to get thinner or was not as stable as in wild type (Fig. 3B) but germ cells did not loose contacts yet and gonads kept their tubular shape, similarly to wild-type (Fig. 3A). At the L3 stage, there was no laminin-GFP detectable in the center of the mutant gonad and the germ cells were released. Gonad disruption appeared always on the dorsal side (Fig. 3D). During gonad isolation in the adult worms we observed that gut and gonad were often joined together in ten-1 mutants suggesting that the regional deficiency in part of the gonadal basement membrane led to germ cell adhesion to the gut.
Local removal of gonadal basement membrane is known to take place during anchor cell invasion. In wild type worms this occurs on the ventral side of the gonad (Fig. 3E), while the break in the ten-1 mutant appeared always on the dorsal side. Since ten-1 is highly expressed in the anchor cell, we considered the possibility that the protein was required for anchor cell formation, guidance or correct spatial attachment and that the anchor cell
localization of the anchor cell in ten-1 mutant worms expressing GFP under the ten-1a promoter. We did not find any defects in the position of the anchor cell in the mutant worms and it attached normally to the ventral side of the gonadal basement membrane at the L3 stage (Fig. 3F).
We also analyzed the gonadal basement membrane ultrastructure by electron microscopy and did not find any obvious general defects in its organization (unpublished data). The basement membrane was absent in the region of the break but appeared normal in the distal parts of the gonad. Moreover, we did not find any whorls or clumps of extracellular material typical for some other basement membrane mutants such as epi-1, lam-1 or dig-1 (Benard et al., 2006; Huang et al., 2003; Kao et al., 2006).
In summary, the gonadal basement membrane in the ten-1(ok641) hermaphrodites was properly assembled at hatching but was not maintained later in development. The localized basement membrane deficiency was not the result of impaired anchor cell invasion but must be due to another cause resulting in defects in the basement membrane assembly, stability or protein expression. Basement membranes beyond the gonad (and pharynx) did not show any major changes in the ten-1 mutant worms as examined with LAM-1::GFP marker (unpublished data).
Gonadal defects of ten-1 mutants are similar to those found in the dystroglycan dgn- 1, integrin ina-1 and laminin epi-1 mutants
Gonadal epithelialization defects were reported for the dystroglycan dgn-1(cg121) mutant (Johnson et al., 2006), several integrin α chain mutants ina-1 (Baum and Garriga, 1997) and laminin α chain mutants epi-1 (Huang et al., 2003). Dystroglycan and integrins are cell surface receptors that interact with laminin and are required for basement membrane assembly, adhesion and signal transduction (Bokel and Brown, 2002; Higginson and Winder, 2005). EPI-1 is one of two laminin α chains found in C. elegans genome.
Laminins are secreted proteins that play fundamental roles in basement membrane formation and function (Miner and Yurchenco, 2004; Previtali et al., 2003). Both C.
elegans laminin isoforms are broadly distributed among the basement membranes but the
RESULTS - SUBMITTED
development and led to worm sterility. Early gonads of ina-1(gm39) worms hardly ever burst (Fig. 4F) and rather seemed to be swollen in the center. However, at the L4 stage ina-1 mutant gonads were clearly ruptured and the germ cells clustered around the developing vulva (Fig. 4G), similarly to ten-1(ok641) gonads (Fig. 4H).
We analyzed the organization of the laminin network surrounding the developing gonad in dgn-1 mutants using LAM-1::GFP marker. Although the DIC pictures of ten-1 and dgn-1 mutants appeared similar, dgn-1(cg121) hermaphrodite gonads did not have any localized break such as the ten-1(ok641) gonads. In contrast, the dgn-1 mutant gonads were generally disorganized and LAM-1::GFP seemed to be more diffuse throughout the gonadal surface in comparison to ten-1 mutant gonads (Fig. 4D). Gonads in the adult dgn-1(cg121) as well as epi-1(RNAi) worms were also more disorganized than in the ten- 1(ok641) mutant (unpublished data).
Nevertheless, gonadal defects described for the above three mutants, i.e. dgn-1, ina-1 and epi-1, resembled the defects that we observed in the ten-1(ok641) worms (Fig. 4B) suggesting that TEN-1 could be an additional receptor required for gonadal basement membrane maintenance.
TEN-1 was found to be expressed in early gonads, including Z1 and Z4 cells, somatic gonad precursor cells during L2 stage (Fig. 4I,J), and anchor cell in L3 stage (Fig. 3E).
TEN-1 expression in these gonadal somatic cells suggested that they could play an important role in the basement membrane maintenance. However, it is unclear whether TEN-1 functions in these cells to control correct basement membrane assembly, somatic cell position in the gonad or cell adhesion.
ten-1 is synthetic lethal with dgn-1, ina-1, epi-1 and nid-1
The similar gonadal phenotypes of ten-1, dgn-1, ina-1 and epi-1 mutants suggested that TEN-1 could be a basement membrane receptor with similar and partly redundant function to dystroglycan and/or integrin receptors. To asses the interaction between ten-1 and genes encoding various basement membrane components, we constructed double mutant combinations. In the crosses we used ten-1(ok641) and dgn-1(cg121) null alleles, the ina-1(gm144) loss-of-function mutant, and an RNAi approach in the case of epi-1.
